Microenvironmental determinants of glioma cell behavior are incompletely understood. The age and neuroanatomical location predilections of glioma occurrence indicate important interactions between the cell of origin and its microenvironment and suggest dysregulation of mechanisms of neurodevelopment and/or plasticity. We recently showed active neurons exert a mitogenic effect on normal neural precursor and oligodendroglial precursor cells, the putative cellular origins for high-grade glioma (HGG). We now preliminarily demonstrate that active neurons similarly promote HGG proliferation in vivo using optogenetic control of cortical neuronal activity in a patient-derived pediatric glioblastoma orthotopic xenograft model. Activity-regulated mitogen(s) are secreted, as the conditioned medium from optogenetically stimulated cortical slices promoted proliferation of pediatric and adult patient-derived HGG cultures. The synaptic protein neuroligin-3 (NLGN3) was identified as the leading candidate mitogen; soluble NLGN3 was sufficient and necessary to promote robust HGG cell proliferation. NLGN3 induced PI3K pathway activity and feed-forward expression of NLGN3 in glioma cells, providing mechanistic insight into its surprising role as a mitogen. NLGN3 expression levels in human HGG negatively correlated with patient overall survival. These findings indicate the important role of active neurons in the brain tumor microenvironment and identify secreted neuroligin-3 as an unexpected mechanism promoting neuronal activity-regulated cancer growth. The proposed work aims to investigate further the direct interaction between NLNG3 and high-grade glioma cells. We will directly probe the necessity of NLGN3 in neuronal activity regulated glioma growth in an in vivo optogenetic model. Further, the research will identify mechanisms of secretion, receptor binding, and downstream signaling pathways. The proposed work highlights the previously under-recognized importance of neuronal activity in the glioma microenvironment, will result in a better mechanistic understanding of the newly identified glioma mitogen NLGN3, and may identify novel therapeutic targets to better treat these deadly brain tumors.